Bottom Line:
Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined.Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities.Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes.

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined.

Methodology/principal findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca²⁺ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca²⁺ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes.

Conclusions/significance: Since Ca²⁺ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract.

pone-0032354-g004: Tas1r1 expression in mammalian spermatozoa.[A] Extrusion of the mCherry protein during spermmaturation in the epididymis. Cryosections of the caput of theepididymis of a Tas1r1/mCherry reporter mouse were incubated with ananti-mCherry antiserum (red; [mCherry]) andcounterstained with the nuclear dye TO-PRO-3 (blue;[TOPRO]).([mCherry+TOPRO], inset, arrowhead).[B] mCherry fluorescence is not detectable inmature epididymal sperm. Isolated sperm of the mutant mouse line werefixed with PFA and counterstained with the FITC-coupled acrosomal markerPNA (middle panel; arrow; [PNA]). Imagingsperm for mCherry fluorescence revealed that the fluorescent protein wascompletely lost during epididymal maturation (left panel[mCherry]). Insets in the right panelsshow higher magnification of the tubule's lumen[A] or a sperm's acrosome[B], respectively. [C]Expression of Tas1r1 in human spermatozoa. Ejaculated human sperm wereincubated with a human specific Tas1r1 antiserum; bound primaryantiserum was visualized applying a FITC-conjugated anti-rabbit IgG. Thetwo representative confocal micrographs document that the anti-Tas1r1IgG ([Tas1r1]) showed a staining in theflagellum (arrow) and in the post-acrosomal region as well as at theequatorial segment (arrowheads). Immunostaining in both subcellularcompartments was extinguished upon neutralizing the primary antiserumwith an excess of the corresponding immunogenic peptide (lower panels;[Tas1r1+BP]), thus confirmingspecificity of the detected immunolabeling. Negative controls, in whichthe primary antiserum was omitted, did not show any labeling (data notshown). Confocal images were produced by an overlay of correspondingfluorescence channels (propidium iodide, [red];FITC-conjugated secondary antiserum, [green]) and thetransmission channel. Boxes indicate regions that are magnified ininsets in the right panels. Experiments were repeated with at leastthree independent sperm preparations from different donors, showingcomparable results.

Mentions:
To confirm our immunocytochemical results of Tas1 receptor expression in maturespermatozoa, we monitored the expression of mCherry in reproductive tissues.Therefore, testis sections were prepared from Tas1r1 mCherry knock-in mice andimaged for color-coded cells (Fig.2B). Spermatogenesisis is characterized by a series of mitoticdivisions with distinct stages of differentiating germ cells localized todefined concentric bands of the seminiferous tubules (s. Fig. 3B; schematic drawing in the left panelin the top): Spermatogonia are located in the basal cell layer, followed by twomeiotic spermatocyte division stages and finally haploid spermatids accumulatingin the central cell layer of the tubular unit (for review see [48]). Due tothis defined spatial organization, mCherry fluorescence signals in testiculartissue sections allow to determine at which developmental stages the receptor isexpressed. Moreover, performing combined immunostaining approaches, it isfeasible to simultaneously investigate the spatial expression profile of thetongue-specific dimerization partner of Tas1r1, Tas1r3, in spermatozoa (s. Fig. 2A and B). mCherryfluorescence signals were found in all analyzed seminiferous tubules of crosssections of testicular tissue of the Tas1r1 reporter mouse strain (s. overviewin Fig. 3B; top panel on theright). Comparing mCherry appearance in single tubules, which typically displayone of twelve characteristic combinations of distinct phases of differentiatinggerm cells [49], fluorescence signals were always detected in moremature round and elongated spermatids in the tubular lumen, whereas sparsefluorescence was detected in the periphery, where the early stages ofspermatogenesis occur (Fig.3B). Tas1r3 immunoreactivity was visible in all tubules examined(Fig. 3B; middle andbottom panels, [i], [ii],[iii]). Moreover, we observed that the Tas1r3receptor emerges at the same phases of spermatogenesis as the Tas1r1 reporterprotein: Tas1r3-derived FITC-labeling was most prominent in cells of the luminallayers of the tubular units, where late spermatocytes and spermatids areconcentrated, while no obvious staining was observed in spermatogonia and earlyspermatocytes located in the outer tubule regions (Fig. 3B; upper right panel;[mCherry+Tas1r3M]). At higher magnification,one can observe that the sub-cellular fluorescence of Tas1r3 did not overlapexactly with the fluorescence pattern of mCherry: While the mCherry signalcannot be attributed to a distinct sub-cellular compartment of developing germcells, Tas1r3 staining was mainly concentrated in the developing acrosomalregion of spermatids (Fig.3B; middle and bottom panels; [i], [iii];arrowhead). Since mCherry fluorescence mirrors Tas1r1 promoter activity, thepattern of mCherry labeling might differ from the endogenously expressedreceptor protein. However, one may speculate that the artificial and dispensablemCherry protein gets lost in fully developed germ cells. Recent studies showedthat final steps of spermatogenesis are accompanied by an extensive extrusion ofsuperfluous cytoplasmic components, which are deposited in detachedmembrane-limited organelles, subdivided into small compartments designated asresidual bodies [50] or larger cytoplasmic droplets [51]. To assess mCherry labelingin late stages, its expression was determined in isolated epididymal sperm ofTas1r1/mCherry knock-in animals (Fig. 4B) and in the epididymis (Fig. 4A), the storage organ of maturespermatozoa [51], respectively. Utilizing the DNA-staining dyeTO-PRO-3 ([TOPRO]), we detected nucleus-derivedfluorescent signals in cells lining the epididymal epithelium and in the lumenof the tubules where mature sperm are located (Fig. 4A; [TOPRO]).Thus, Tas1r1 mutant mice show no obvious morphological defects in theepididymis. However, the luminal mCherry immunoreactivity appears to beaccumulated in large vesicular structures most likely representing cellularextrusion organelles (Fig.4A; [mCherry+TOPRO], arrowhead). Extrusion of thecytoplasmatic mCherry protein was confirmed by monitoring mCherry fluorescencein isolated sperm cells: Whereas the lectin PNA (green fluorescence) labeled atypical crescent-shaped acrosome in Tas1r1 mutant sperm (Fig. 4A; arrowhead;[PNA]), red coloration reflecting the presence ofmCherry was not found, even after increasing the sensitivity for mCherrydetection by applying an anti-DsRed antiserum (data not shown). Thus, themCherry fluorescence protein mostly likely represents cellular detritus for germcells and might be excluded from maturing spermatozoa. However, since it iswidely accepted that sperm are transcriptionally and translationally silent[52],proteins essential for a successful fertilization already have to be synthesizedduring sperm cell development. Therefore, the marked increase in mCherryfluorescence intensity at late stages of spermatogenesis (Fig. 3B) together with the co-localization ofits obligatory dimerization partner, the Tas1r3 protein, in mature spermatozoa(Fig. 2B), can reliablybe interpreted to indicate the presence of the Tas1r1 receptor protein in fullydeveloped germ cells. However, due to the shortcomings of commercially availableantibodies, we were unable to confirm the expression of the Tas1r1 protein inmature sperm, at least in mouse. Due to the availability of reliable functioningantisera against the human Tas1r1 receptor protein, we decided to clarify thispoint in human sperm cells. To validate the specificity of Tas1r1 antisera ofwhich four had been reported to detect the human umami taste receptor, wetransfected HEK293 cells with a human Tas1r1 cDNA fused to a Herpes SimplexVirus (HSV)-tag. In Western blot experiments we found that one testedanti-Tas1r1 IgG (Tas1r1 A, Acris) detected a single immuno-reactive band withthe expected size of the Tas1r1 (93 kDa) (Fig. S2A;[ab]). This band was also labeled with ananti-HSV antiserum (data not shown) and was eliminated by preincubation with theimmunogenic peptide (Fig. S2A,[ab+bp]). This Tas1r1 antiserum was subsequentlyused to analyze umami taste receptor expression in freshly ejaculated humansperm. The antiserum caused immunostaining of human sperm (for representativeresults s. Fig. 4C;[Tas1r1]) that was abolished after neutralizingwith the antigenic peptide (Fig.4C; bottom panel [Tas1r1+BP]). Athigher magnification, staining was detected in both subcellular compartments ofthis germ cell type: Labeling of the sperm flagellum was most prominent in themitochondria-rich mid-piece segment (upper panel in Fig. 4C; arrows) whereas the flagellum'sprincipal and end tail segments only showed faint immunoreactivity. In addition,the post-acrosomal region and the equatorial segment of the paddle-shaped headwere labeled (Fig. 4B;higher magnifications in the right panels; arrowhead). Of note, immunostainingof the potential dimerization partner of Tas1r1 in human sperm, using anantiserum which also specifically labeled the recombinant protein in HEK cells(Fig.S2B) [53], revealed a comparable, but slightly broadersubcellular expression pattern which also encompassed the acrosomal cap and thesperm flagellum (Fig. S2C). These observations indicate that the two subunits formingthe tongue umami taste receptor show an overlapping subcellular distributionpattern in sperm of different mammalian species.

pone-0032354-g004: Tas1r1 expression in mammalian spermatozoa.[A] Extrusion of the mCherry protein during spermmaturation in the epididymis. Cryosections of the caput of theepididymis of a Tas1r1/mCherry reporter mouse were incubated with ananti-mCherry antiserum (red; [mCherry]) andcounterstained with the nuclear dye TO-PRO-3 (blue;[TOPRO]).([mCherry+TOPRO], inset, arrowhead).[B] mCherry fluorescence is not detectable inmature epididymal sperm. Isolated sperm of the mutant mouse line werefixed with PFA and counterstained with the FITC-coupled acrosomal markerPNA (middle panel; arrow; [PNA]). Imagingsperm for mCherry fluorescence revealed that the fluorescent protein wascompletely lost during epididymal maturation (left panel[mCherry]). Insets in the right panelsshow higher magnification of the tubule's lumen[A] or a sperm's acrosome[B], respectively. [C]Expression of Tas1r1 in human spermatozoa. Ejaculated human sperm wereincubated with a human specific Tas1r1 antiserum; bound primaryantiserum was visualized applying a FITC-conjugated anti-rabbit IgG. Thetwo representative confocal micrographs document that the anti-Tas1r1IgG ([Tas1r1]) showed a staining in theflagellum (arrow) and in the post-acrosomal region as well as at theequatorial segment (arrowheads). Immunostaining in both subcellularcompartments was extinguished upon neutralizing the primary antiserumwith an excess of the corresponding immunogenic peptide (lower panels;[Tas1r1+BP]), thus confirmingspecificity of the detected immunolabeling. Negative controls, in whichthe primary antiserum was omitted, did not show any labeling (data notshown). Confocal images were produced by an overlay of correspondingfluorescence channels (propidium iodide, [red];FITC-conjugated secondary antiserum, [green]) and thetransmission channel. Boxes indicate regions that are magnified ininsets in the right panels. Experiments were repeated with at leastthree independent sperm preparations from different donors, showingcomparable results.

Mentions:
To confirm our immunocytochemical results of Tas1 receptor expression in maturespermatozoa, we monitored the expression of mCherry in reproductive tissues.Therefore, testis sections were prepared from Tas1r1 mCherry knock-in mice andimaged for color-coded cells (Fig.2B). Spermatogenesisis is characterized by a series of mitoticdivisions with distinct stages of differentiating germ cells localized todefined concentric bands of the seminiferous tubules (s. Fig. 3B; schematic drawing in the left panelin the top): Spermatogonia are located in the basal cell layer, followed by twomeiotic spermatocyte division stages and finally haploid spermatids accumulatingin the central cell layer of the tubular unit (for review see [48]). Due tothis defined spatial organization, mCherry fluorescence signals in testiculartissue sections allow to determine at which developmental stages the receptor isexpressed. Moreover, performing combined immunostaining approaches, it isfeasible to simultaneously investigate the spatial expression profile of thetongue-specific dimerization partner of Tas1r1, Tas1r3, in spermatozoa (s. Fig. 2A and B). mCherryfluorescence signals were found in all analyzed seminiferous tubules of crosssections of testicular tissue of the Tas1r1 reporter mouse strain (s. overviewin Fig. 3B; top panel on theright). Comparing mCherry appearance in single tubules, which typically displayone of twelve characteristic combinations of distinct phases of differentiatinggerm cells [49], fluorescence signals were always detected in moremature round and elongated spermatids in the tubular lumen, whereas sparsefluorescence was detected in the periphery, where the early stages ofspermatogenesis occur (Fig.3B). Tas1r3 immunoreactivity was visible in all tubules examined(Fig. 3B; middle andbottom panels, [i], [ii],[iii]). Moreover, we observed that the Tas1r3receptor emerges at the same phases of spermatogenesis as the Tas1r1 reporterprotein: Tas1r3-derived FITC-labeling was most prominent in cells of the luminallayers of the tubular units, where late spermatocytes and spermatids areconcentrated, while no obvious staining was observed in spermatogonia and earlyspermatocytes located in the outer tubule regions (Fig. 3B; upper right panel;[mCherry+Tas1r3M]). At higher magnification,one can observe that the sub-cellular fluorescence of Tas1r3 did not overlapexactly with the fluorescence pattern of mCherry: While the mCherry signalcannot be attributed to a distinct sub-cellular compartment of developing germcells, Tas1r3 staining was mainly concentrated in the developing acrosomalregion of spermatids (Fig.3B; middle and bottom panels; [i], [iii];arrowhead). Since mCherry fluorescence mirrors Tas1r1 promoter activity, thepattern of mCherry labeling might differ from the endogenously expressedreceptor protein. However, one may speculate that the artificial and dispensablemCherry protein gets lost in fully developed germ cells. Recent studies showedthat final steps of spermatogenesis are accompanied by an extensive extrusion ofsuperfluous cytoplasmic components, which are deposited in detachedmembrane-limited organelles, subdivided into small compartments designated asresidual bodies [50] or larger cytoplasmic droplets [51]. To assess mCherry labelingin late stages, its expression was determined in isolated epididymal sperm ofTas1r1/mCherry knock-in animals (Fig. 4B) and in the epididymis (Fig. 4A), the storage organ of maturespermatozoa [51], respectively. Utilizing the DNA-staining dyeTO-PRO-3 ([TOPRO]), we detected nucleus-derivedfluorescent signals in cells lining the epididymal epithelium and in the lumenof the tubules where mature sperm are located (Fig. 4A; [TOPRO]).Thus, Tas1r1 mutant mice show no obvious morphological defects in theepididymis. However, the luminal mCherry immunoreactivity appears to beaccumulated in large vesicular structures most likely representing cellularextrusion organelles (Fig.4A; [mCherry+TOPRO], arrowhead). Extrusion of thecytoplasmatic mCherry protein was confirmed by monitoring mCherry fluorescencein isolated sperm cells: Whereas the lectin PNA (green fluorescence) labeled atypical crescent-shaped acrosome in Tas1r1 mutant sperm (Fig. 4A; arrowhead;[PNA]), red coloration reflecting the presence ofmCherry was not found, even after increasing the sensitivity for mCherrydetection by applying an anti-DsRed antiserum (data not shown). Thus, themCherry fluorescence protein mostly likely represents cellular detritus for germcells and might be excluded from maturing spermatozoa. However, since it iswidely accepted that sperm are transcriptionally and translationally silent[52],proteins essential for a successful fertilization already have to be synthesizedduring sperm cell development. Therefore, the marked increase in mCherryfluorescence intensity at late stages of spermatogenesis (Fig. 3B) together with the co-localization ofits obligatory dimerization partner, the Tas1r3 protein, in mature spermatozoa(Fig. 2B), can reliablybe interpreted to indicate the presence of the Tas1r1 receptor protein in fullydeveloped germ cells. However, due to the shortcomings of commercially availableantibodies, we were unable to confirm the expression of the Tas1r1 protein inmature sperm, at least in mouse. Due to the availability of reliable functioningantisera against the human Tas1r1 receptor protein, we decided to clarify thispoint in human sperm cells. To validate the specificity of Tas1r1 antisera ofwhich four had been reported to detect the human umami taste receptor, wetransfected HEK293 cells with a human Tas1r1 cDNA fused to a Herpes SimplexVirus (HSV)-tag. In Western blot experiments we found that one testedanti-Tas1r1 IgG (Tas1r1 A, Acris) detected a single immuno-reactive band withthe expected size of the Tas1r1 (93 kDa) (Fig. S2A;[ab]). This band was also labeled with ananti-HSV antiserum (data not shown) and was eliminated by preincubation with theimmunogenic peptide (Fig. S2A,[ab+bp]). This Tas1r1 antiserum was subsequentlyused to analyze umami taste receptor expression in freshly ejaculated humansperm. The antiserum caused immunostaining of human sperm (for representativeresults s. Fig. 4C;[Tas1r1]) that was abolished after neutralizingwith the antigenic peptide (Fig.4C; bottom panel [Tas1r1+BP]). Athigher magnification, staining was detected in both subcellular compartments ofthis germ cell type: Labeling of the sperm flagellum was most prominent in themitochondria-rich mid-piece segment (upper panel in Fig. 4C; arrows) whereas the flagellum'sprincipal and end tail segments only showed faint immunoreactivity. In addition,the post-acrosomal region and the equatorial segment of the paddle-shaped headwere labeled (Fig. 4B;higher magnifications in the right panels; arrowhead). Of note, immunostainingof the potential dimerization partner of Tas1r1 in human sperm, using anantiserum which also specifically labeled the recombinant protein in HEK cells(Fig.S2B) [53], revealed a comparable, but slightly broadersubcellular expression pattern which also encompassed the acrosomal cap and thesperm flagellum (Fig. S2C). These observations indicate that the two subunits formingthe tongue umami taste receptor show an overlapping subcellular distributionpattern in sperm of different mammalian species.

Bottom Line:
Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined.Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities.Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes.

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined.

Methodology/principal findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca²⁺ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca²⁺ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes.

Conclusions/significance: Since Ca²⁺ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract.